The 26S protease is a large multisubunit enzyme that degrades important regulatory proteins such as p53, cyclins, NFkappaB and ornithine decarboxylase. It is comprised of a regulatory complex containing at least 15 different subunits associated with the multicatalytic protease which contributes an additional 14 subunits. PCR-based strategies will be used to obtain cDNAs encoding four 26S protease subunits (S2, S9, S11 and S15) that remain unidentified. The cDNAs will be expressed in E. coli, and subunit specific antibodies will be generated from the recombinant proteins. Pulse-chase metabolic labeling and several immunoprecipitation approaches will be used to determine whether components of the 26S protease are in dynamic equilibrium. Antibodies specific to 26S protease subunits (S4, S5a, S5b, S6 and S12) are available. These antibodies and others generated in the near future will be used to determine the tissue distribution and intracellular location of the respective 26S subunits. In two separate projects, site-directed mutagenesis will be used to assess the importance of N-terminal coiled-coil regions of S4-like ATPases (subunits 4, 6, 7 and 8) in assembly of the 26S protease and in substrate selection. Direct filter binding assays will be used to identify proteins that interact with subunit 4. Deletional analysis of the ubiquitin- conjugate binding subunit (S5a) will be employed to determine which region(s) of the protein bind Ub tetramers and to determine how many binding sites are present. Finally, site-directed mutagenesis will be used to assess the importance of specific residues in the tetramer binding regions of S5a. The 26S protease is clearly involved in control of the cell cycle, and it is the enzyme most likely to generate antigenic peptides displayed on MHC Class l molecules. For these reasons, better understanding of the protease should have significant medical implications.